Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Reproduction toxicity (OECDTG 416): NOAEL 165 mg/kg bw/day in males [3000 ppm (corresponding to 165 -237 mg/kg bw/day in males and 232 -499 mg/kg bw/day in females)] (read across from substance p-TSA).

Link to relevant study records

Referenceopen allclose all

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 October 2006 to 02 July 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to OECD and EU guidelines and according to GLP principles.
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.35 (Two-Generation Reproduction Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
(WI) BR (outbred, SPFQuality). Untreated animals and virgin females were used at initiation of the study. This species and strain of rat has been recognized as appropriate
for reproduction studies.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: (P) 5-6 wks; (F1) 3 wks
- Housing: Pre-mating: Animals were housed in groups of 4 animals/sex/cage in Macrolon plastic cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages
Post-mating: Males were housed in groups of 4 animals/sex/cage in Macrolon plastic cages. Females were individually housed in Macrolon plastic cages.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.6-24.2
- Humidity (%): 27-95
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Diets were prepared for one week during the first week of study and every two weeks from Week 2 of study onwards. For the last weeks of the study, diets were prepared for a maximum of 36 days.
- Mixing appropriate amounts with Standard powder rodent diet
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 10 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged: individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Sampling and analysis of diet preparations were performed on four occasions (week 1, 11, 22 and 33) according to the following scheme:
Group 1: accuracy (middle position of container)
Group 2: accuracy and homogeneity (top, middle and bottom position of container)
Group 3: accuracy (middle position of container)
Group 4: accuracy and homogeneity (top, middle and bottom position of container)
Duration of treatment / exposure:
F0-generation: 10 weeks prior to mating and continuing until euthanasia.
F1-generation (F0-offspring): The F1-generation was potentially exposed to the test substance in utero, through nursing during lactation and directly following weaning. After weaning, pups were treated for a minimum of 10 weeks prior to mating and continuing until euthanasia.
F2-generation (F1-offspring): The F2-generation was potentially exposed to the test substance in utero and through nursing during lactation.
Frequency of treatment:
Ad libitum
Details on study schedule:
- F1 parental animals not mated until 70 days after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
Dose / conc.:
1 000 ppm (nominal)
Remarks:
corresponds to 52-78 mg/kg bw/day for males and 75-161 mg/kg bw/day for females,
Dose / conc.:
3 000 ppm (nominal)
Remarks:
corresponds to 165-237 mg/kg bw/day for males and 232-499 mg/kg bw/day for females
Dose / conc.:
10 000 ppm (nominal)
Remarks:
corresponds to 566-832 mg/kg bw/day for males and 733-1631 mg/kg bw/day for females
No. of animals per sex per dose:
24
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: based on results of a 28-day toxicity study in Wistar Han rats at dose levels of 0, 1000, 5000 and 25000 ppm.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily.
- Cage side observations: mortality, viability.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on days 0, 4, 7, 11, 14, 17 and 20 of gestation, and during lactation on days 1, 4, 7, 14 and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION : Yes
- Time schedule for examinations: subjective appraisal.
Oestrous cyclicity (parental animals):
Daily over a period of 3 weeks prior to pairing and throughout cohabitation
Sperm parameters (parental animals):
testis weight, epididymis weight, enumeration of homogenisation-resistant spermatids in testes and epididymis, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology.
Litter observations:
PARAMETERS EXAMINED
number and sex of pups
stillbirths
live births
presence of gross anomalies
weight gain
physical or behavioural abnormalities
The day of vaginal opening or balanopreputial separation for F1-weanlings selected for mating. As a slightly delayed balanopreputial separation and vaginal opening for high dose F1-weanlings was observed, ano-genital distance was measured on day 1 of lactation for all F2-pups.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals as soon as possible after the last litters in each generation were produced.
- Maternal animals: All surviving animals on day 21 post partum or shortly thereafter. Females showing no evidence of copulation were killed approximately 21 days after the last day of the mating period.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were prepared for microscopic examination: cervix, coagulation gland, epididymides, ovaries, prostate gland, seminal vesicles, testes, uterus, vagina, all gross lesions.
The following organs were weighed: Adrenal glands, Brain, Epididymides, Kidneys, Liver, Ovaries, Pituitary gland, Prostate, Seminal vesicles, Spleen,
Testes, Thyroid, Uterus

Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at day 21 post partum or shortly thereafter.
- These animals were subjected to postmortem examinations (macroscopic and microscopic examination) as follows:
Vagina, uterus, ovaries, testis, epididymis, seminal vesicle, prostate, coagulating gland

GROSS NECROPSY
- Gross necropsy consisted of external examination of the cranium, and macroscopic examination of the thoracic and abdominal tissues and organs with emphasis on developmental morphology.

HISTOPATHOLOGY / ORGAN WEIGTHS
The following tissues were prepared for microscopic examination: Vagina, uterus, ovaries, testis, epididymis, seminal vesicle, prostate, coagulating gland
The following tissues were weighed: brain, spleen, thymus
Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Fisher 1950) was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 were accepted as the lowest level of significance.
Reproductive indices:
Percentage mating: Number of females mated x 100 / Number of females paired
Fertility index: Number of pregnant females x 100 / Number of females paired
Conception rate: Number of pregnant females x 100 / Number of females mated
Gestation index: Number of females bearing live pups x 100 / Number of pregnant females
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
Percentage live males at First Litter Check: Number of live male pups at First Litter Check x 100 / Number of live pups at First Litter Check
Percentage live females at First Litter Check: Number of live female pups at First Litter Check x 100 / Number of live pups at First Litter Check
Percentage of postnatal loss days 0-4 post partum: Number of dead pups on day 4 post partum x 100 / Number of live pups at First Litter Check
Percentage of breeding loss day 5 until weaning: Number of dead pups between days 5 and 21 post partum x 100 / Number of live pups on day 4 post partum
Percentage live males at weaning: Number of live male pups on day 21 post partum x 100 / Number of live pups on day 21 post partum
Percentage live females at weaning: Number of live female pups on day 21 post partum x 100 / Number of live pups on day 21 post partum
Viability index: Number of live pups on day 4 post partum x 100 / Number of pups born alive
Weaning index: Number of live pups on day 21 post partum x 100 / Number of live pups on day 4 post partum
Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
ORGAN WEIGHTS (PARENTAL ANIMALS)
Males P generation:
At 3000 ppm:
Decreased body weights and body weight gain. Terminal body weights at necropsy (92% of control) were decreased which resulted in relative organ weight changes for the brain and kidneys (105% and 110% of control, respectively).
At 10000 ppm:
Decreased body weights and body weight gain. Terminal body weights at necropsy (91% of control) were decreased which resulted in relative organ weight changes for the brain, liver, kidneys and seminal vesicles (108%, 108%, 118% and 116% of control, respectively).

Females P generation:
At 3000 ppm:
Decreased body weights and body weight gain. Terminal body weights at necropsy (94% of control) were decreased which resulted in relative organ weight changes for the brain (107% of control).
At 10000 ppm:
Decreased body weights and body weight gain. Terminal body weights at necropsy (91% of control) were decreased which resulted in relative organ weight changes for the brain, kidneys, adrenals and spleen (109%, 121%, 112% and 110% of control, respectively).

Key result
Dose descriptor:
NOAEL
Remarks:
parental
Effect level:
1 000 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: Corresponds to 52-78 (average 65) mg/kg bw/day for males and 75-161 mg/kg bw/day for females.
Remarks on result:
other: Generation: P and F1 (migrated information)
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction and breeding
Effect level:
10 000 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: Corresponds to 566-832 mg/kg bw/day for males and 733-1631 mg/kg bw/day for females.
Remarks on result:
other: Generation: P and F1 (migrated information)
Dose descriptor:
NOAEL
Remarks:
developmental
Effect level:
3 000 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: Corresponds to 165-237 mg/kg bw/day for males and 232-499 mg/kg bw/day for females.
Remarks on result:
other: Generation: F1 and F2 (migrated information)
Dose descriptor:
LOAEL
Remarks:
parental
Effect level:
3 000 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: Based on decreased body weights and body weight gain, and due to this organ weight changes.
Remarks on result:
other: Generation: P and F1 (migrated information)
Dose descriptor:
LOAEL
Remarks:
developmental
Effect level:
10 000 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: Based on decreased body weights and body weight gain, and due to this organ weight changes.
Remarks on result:
other: Generation: F1 and F2 (migrated information)
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Males F1 generation:
At 3000 ppm:
Decreased body weights and body weight gain. Terminal body weights at necropsy (93% of control) were decreased which resulted in absolute organ weight changes for the spleen (90% of control) and relative organ weight changes for the brain (108% of control).
At 10000 ppm:
Decreased body weights and body weight gain. Terminal body weights at necropsy (87% of control) were decreased which resulted in absolute organ weight changes for the brain, liver, and spleen (95%, 89% and 89% of control, respectively), and relative organ weight changes for the brain, kidneys, seminal vesicles, testes and prostate (108%, 111%, 124%, 109% and 118% of control, respectively).

Females F1 generation:
At 3000 ppm:
Decreased body weights, body weight gain and terminal body weights at necropsy (93% of control).
At 10000 ppm:
Decreased body weights and body weight gain. Decreased food consumption during the last week of pregnancy. Terminal body weights at necropsy (85% of control) were decreased which resulted in absolute organ weight changes for the brain (93% of control) and relative organ weight changes for the brain, kidneys, adrenals, spleen and pituitary gland (110%, 118%, 112%, 113% and 120% of control, respectively).


BODY WEIGHT (OFFSPRING)
ORGAN WEIGHTS (OFFSPRING)
Male pups F1 generation:
At 10000 ppm:
Lower body weights for pups (terminal body weight at necropsy was 85% of control) which resulted in absolute organ weight changes for the brain, spleen and thymus (95%, 78% and 74% of control, respectively), relative organ weight changes for the brain (111% of control), and a delay in balanopreputial separation (108% of control).

Female pups F1 generation:
At 10000 ppm:
Lower body weights for pups (terminal body weight at necropsy was 84% of control) which resulted in absolute organ weight changes for the brain, spleen and thymus (94%, 78% and 73% of control, respectively), relative organ weight changes for the brain (112% of control), and a delay in vaginal opening (114% of control).

Key result
Dose descriptor:
NOAEL
Remarks:
parental
Generation:
F1
Effect level:
1 000 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: Corresponds to 52-78 (average 65) mg/kg bw/day for males and 75-161 mg/kg bw/day for females.
Remarks on result:
other: Generation: P and F1 (migrated information)
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction and breeding
Generation:
F1
Effect level:
10 000 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: Corresponds to 566-832 mg/kg bw/day for males and 733-1631 mg/kg bw/day for females.
Remarks on result:
other: Generation: P and F1 (migrated information)
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
3 000 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: Corresponds to 165-237 mg/kg bw/day for males and 232-499 mg/kg bw/day for females.
Remarks on result:
other: Generation: F1 and F2 (migrated information)
Dose descriptor:
LOAEL
Remarks:
parental
Generation:
F1
Effect level:
3 000 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: Based on decreased body weights and body weight gain, and due to this organ weight changes.
Remarks on result:
other: Generation: P and F1 (migrated information)
Dose descriptor:
LOAEL
Remarks:
developmental
Generation:
F1
Effect level:
10 000 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: Based on decreased body weights and body weight gain, and due to this organ weight changes.
Remarks on result:
other: Generation: F1 and F2 (migrated information)
BODY WEIGHT (OFFSPRING)
ORGAN WEIGHTS (OFFSPRING)

Male pups F2 generation:
At 10000 ppm:
Lower body weights for pups (terminal body weight at necropsy was 81% of control) which resulted in absolute organ weight changes for the brain, spleen and thymus (95%, 75% and 70% of control, respectively) and relative organ weight changes for the brain (117% of control).

Female pups F2 generation:
At 10000 ppm:
Lower body weights for pups (terminal body weight at necropsy was 82% of control) which resulted in absolute organ weight changes for the spleen and thymus (76% and 73% of control, respectively) and relative organ weight changes for the brain and thymus (118% and 90% of control, respectively).
Reproductive effects observed:
not specified

Table 1. Mean test article intake when corrected for mean value of recovery

Nominal dose

1000 ppm

3000 ppm

10000 ppm

Mean value of recovery

930 ppm

2820 ppm

9600 ppm

MALES F0

Pre mating

75

236

787

Post mating

52

165

568

FEMALES F0

Premating

85

264

862

Postcoitum

75

238

739

Lactation

161

499

1631

MALES F1

Premating

78

237

832

Post mating

53

165

566

FEMALES F1

Premating

86

264

887

Postcoitum

75

232

733

Lactation

160

487

1582

Table 2. Reproductive toxicity

Dose (ppm)

0

1000

3000

10000

dr

m

f

m

f

m

f

m

f

F0 animals

Mortality

no treatment-related findings

Clinical signs

no treatment-related findings

Body weight gain

dc

dc

dc

dc

mf

Food consumption

no treatment-related findings

Mating/fertility/gestation

no treatment-related findings

Oestrus cycle

no treatment-related findings

Sperm evaluation

no treatment-related findings

Organ weight

- terminal body weight

dc (94%)

dc (92%)

dc (91%)

dc (91%)

- brain

icr

(107%)

icr

(105%)

icr

(109%)

icr

(108%)

- liver

icr

(108%)

- kidneys

icr

(110%)

icr

(121%)

icr

(118%)

f

- adrenals

icr

(112%)

- spleen

icr

(110%)

- seminal vesicles

icr

(116%)

Pathology

Macroscopy

no treatment-related findings

Microscopy

no treatment-related findings

F1 pups

Litter size

no treatment-related findings

Post implantation loss

no treatment-related findings

Live birth index

no treatment-related findings

Viability index

no treatment-related findings

Lactation index

no treatment-related findings

Sex ratio

no treatment-related findings

Clinical signs

no treatment-related findings

Body weight

dc

dc

Sexual maturation

ic

ic

Pup development

no treatment-related findings

Organ weight

- terminal body weight

dc (84%)

dc (85%)

- brain

dca (94%)

icr

(112%)

dca (95%)

icr

(111%)

- spleen

dca (78%)

dca (78%)

- thymus

dca (73%)

dca (74%)

Pathology

Macroscopy

no treatment-related findings

Microscopy

no treatment-related findings

F1 animals

Mortality

no treatment-related findings

Clinical signs

no treatment-related findings

Body weight gain

dc

dc

dc

dc

mf

Food consumption

dc

Mating/fertility/gestation

no treatment-related findings

Oestrus cycle

no treatment-related findings

Sperm evaluation

no treatment-related findings

Organ weight

- terminal body weight

dc (93%)

dc (93%)

dc (87%)

dc (85%)

mf

- brain

icr (108%)

dca (95%)

icr (108%)

dca (93%)

icr (110%)

- pituitary

icr (120%)

- liver

dca (89%)

- kidneys

icr (111%)

icr (118%)

- adrenals

icr (112%)

- spleen

dca (90%)

dca (89%)

icr (113%)

- testes

icr (109%)

- prostate

icr (118%)

- seminal vesicles

icr (124%)

Pathology

Macroscopy

no treatment-related findings

Microscopy

no treatment-related findings

F2 pups

Litter size

no treatment-related findings

Post implantation loss

no treatment-related findings

Live birth index

no treatment-related findings

Viability index

no treatment-related findings

Lactation index

no treatment-related findings

Sex ratio

no treatment-related findings

Clinical signs

no treatment-related findings

Body weight

dc

dc

Pup development

no treatment-related findings

Auditory and visual response

not performed

Organ weight

- terminal body weight

dc (81%)

dc (82%)

- brain

dca (95%)

icr

(117%)

icr

(118%)

- spleen

dca (75%)

dca (76%)

- thymus

dca (70%)

dca (73%)

dcr

(90%)

 Pathology

no treatment-related findings

dc/ic     statistically significantly decreased/increased compared to the controls

a/r        absolute/relative organ weight

(%)       relative to control

dr         dose-related

Conclusions:
Parental toxicity was observed for both generations at the mid and high dose groups (3000 and 10000 ppm).
Developmental toxicity was observed for both generations at the high dose group (10000 ppm).
Reproduction and breeding parameters were unaffected for both generations for treatment up to 10000 ppm.

Based on these findings, the definitive parental No Observed Adverse Effect Level (NOAEL) was established as being 1000 ppm.
The definitive development NOAEL was established as being 3000 ppm.
The definitive reproduction and breeding NOAEL was established as being at least 10000 ppm.

When corrected for mean test article intake the NOAEL of 1000 ppm corresponds to 52-78 (average 65) mg/kg bw/day for males and 75-161 mg/kg bw/day for females, the NOAEL of 3000 ppm corresponds to 165-237 mg/kg bw/day for males and 232-499 mg/kg bw/day for females, and the NOAEL of 10000 ppm corresponds to 566-832 mg/kg bw/day for males and 733-1631 mg/kg bw/day for females.
NOAEL parenteral is used as starting point for the DNEL derivation (NOAEL parenteral = 65 mg/kg bw/day);

These results were used for read-across to Tosylchloramide sodium.
Executive summary:

A two-generation reproduction toxicity study of p-TSA in rats by dietary administration.

The study was based on the following guidelines:

- OECD 416, Two-Generation Reproduction Toxicity Study, January 2001.

- OPPTS 870.3800, Reproduction and Fertility Effects, August 1998.

- EC, Commission directive 2004/73/EC, B.35:"Two-generation reproduction toxicity study", April 2004.

 

After acclimatisation, male and female Wistar rats of the Fe-generation were exposed by dietary inclusion to graduated doses of the test substance. The dose levels for the F0-generation and for the F1-generation were 1000, 3000 and 10000 ppm.

At regular intervals, prepared diets were analysed for accuracy of preparation and homogeneity.

F0-males and F0-females were exposed to the test substance 10 weeks prior to mating and exposure ended at termination. F0-females were allowed to produce and rear a litter until day 21 of lactation. On day 4 of lactation litters were reduced in size to eight pups by random culling of F1-pups. After weaning, one F1-male and one F1-female of each litter were selected for cross mating with a pup of another litter of the same dose group to produce a F2-generation. Mating commenced at least 10 weeks after weaning. F1-offspring selected for mating were dosed from weaning until they were killed after weaning of the F2-offspring on day 21 of lactation. On day 4 of lactation a selection of F2-pups was culled. Pups of the F2-offspring were killed shortly after weaning.

 

All animals were subjected to daily clinical observation. Body weight and food consumption were measured over the treatment period. The regularity and duration of the estrous cycle was examined. At necropsy, macroscopic observations and organ weights were recorded. Sperm morphology, motility and count were assessed. A histopathological examination was performed on all reproduction organs and tissues. Reproduction parameters, breeding data and pup development were assessed. Blood samples were collection from F1 females (10/group) for possible measurement of thyroid hormones. These samples were discarded without analyses.

 

RESULTS

Chemical analysis revealed that the diets were prepared properly and were considered to be homogeneous.

 

The following changes were considered to be related to treatment:

F0-GENERATION

at 1000 ppm (group 2):

•      No treatment-related findings.

 

at 3000 ppm (group 3):

•      Decreased body weights and body weight gain. Terminal body weights at necropsy were decreased which resulted in relative organ weight changes for the brain and kidneys.

 

at 10000 ppm (group 4):

•      Decreased body weights and body weight gain. Terminal body weights at necropsy were decreased which resulted in relative organ weight changes for the brain, liver, kidneys, seminal vesicles, adrenals, and spleen. Lower body weights for male and female pups which resulted in organ weight changes for the brain, spleen and thymus and a delay in vaginal opening and balanopreputial separation.

 

F1-GENERATION

at1000ppm (group2):

•      No treatment-related findings.

 

at3000ppm (group3):

•      Decreased body weights and body weight gain. Terminal body weights at necropsy were decreased which resulted in organ weight changes for the brain and spleen.

 

at10000ppm (group4):

•      Decreased body weights and body weight gain. Decreased food consumption for females during the last week of pregnancy. Terminal body weights at necropsy were decreased which resulted in organ weight changes for the brain, liver, kidneys, seminal vesicles, adrenals, spleen, testes, prostate, and pituitary gland. Lower body weights for male and female pups which resulted in organ weight changes for the brain, spleen and thymus.

 

CONCLUSION

Treatment with p-TSA by dietary inclusion in male and female Wistar rats at dose levels of 1000, 3000 and 10000 ppm revealed Fo-and F1-parental toxicity at 3000 and 10000 ppm and developmental toxicity (related to decreased body weights of dams) at 10000 ppm.

Reproduction and breeding parameters were unaffected for both generations for treatment up to 10000 ppm.

 

Based on these findings, the definitive parental No Observed Adverse Effect Level (NOAEL) was established as being 1000 ppm.

The definitive reproduction and breeding NOAEL was established as being at least 10000 ppm.

The definitive development NOAEL was established as being 3000 ppm.

 

When corrected for mean test article intake the

NOAEL of 1000 ppm corresponds to 52 -78 (average 65) mg/kg bw/day for males and 75-161 mg/kg bw/day for females,

NOAEL of 3000 ppm corresponds to 165-237 mg/kg bw/day for males and 232 -499 mg/kg bw/day for females,

NOAEL of 10000 ppm corresponds to 566 -832 mg/kg bw/day for males and 733 -1631 mg/kg bw/day for females.

NOAEL parenteral is used as starting point for the DNEL derivation (NOAEL parenteral = 65 mg/kg bw/day);    

These results were used for read-across to Tosylchloramide Sodium.

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Original report in Japanese, but translation and OECD SIDS reporting are sufficient and trustworthy.
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Sprague-Dawley lineage (Crj:CD, SPF) from Atsugi Breeding Center of Charles River Japan, Inc.
7-week-old male and female
Acclimatisation: 1 week
weight range at the time of grouping: male 186.3-225.0g, female 268.4-306.5g.
Housing: individually in a metal wire netting floor cage (22×27×19mm, Japan Cage Co., Ltd.)
Housing: Room temp: 24±1℃, relative humidity 55±5%, Air changes 15 times/hour, 12-hour lighting
Feed|: Ad libitum; solid feed (CA-1, CLEA Japan, Inc.) and tap water.
For females later than 18 days of pregnancy, a metal plate was placed on the cage floor and wood chips (White Flake (R), Charles River Japan, Inc.) were provided as bedding.
The provided feed, water, and bedding did not include any impurity that may have affected the experiment.
Route of administration:
oral: gavage
Vehicle:
other: 5% solution of Arabic gum
Details on exposure:
The test substance was suspended in a 5% Gum Arabic solution [Gum Arabic: Lot No., JD03 (Miyazawa Chemicals); distilled water for injection: Lot No., L05019 (Yamaguchi Pharmaceutical)].
Dose volume: 5 ml/kg
The prepared administration samples were sealed and stored under cool, dark condition and were administered within 7 days after preparation.
Daily administration; amount administered solution was calculated from weekly weight measurements for males and females before and during mating period, and from the weight at day 0 of pregnancy for females after mating completion.
Details on mating procedure:
- M/F ratio per cage: 1 male and 1 female per cage
- Length of cohabitation: up to max 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Rate of successful mating reported. No further atempt when not succesful
- After successful mating each pregnant female was individual caged
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
We have confirmed that the test substance in the prepared solution was stable under the storage condition of room temperature and without light for at least 7 days.
We also confirmed that the desired amount of 4-methylbenzenesulfonamide was included in each administration specimen.
Duration of treatment / exposure:
Females, from 14 days before mating through mating and pregnancy to day 3 of lactation.
Males 42 days
Frequency of treatment:
Daily administration was done basically during a fixed range of time (normally between 1PM to 4PM),

Dose / conc.:
120 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
13
Control animals:
yes, concurrent vehicle
Details on study design:
Section: Animals were kept away from feed for approximately 18 hours until they were let to bleed to death under deep pentobarbital anesthesia for autopsy on the following day (day 42 of administration). Prior to autopsy for all animals, blood was for haematological and biochemical evaluations collected from post abdominal vena cava under
pentobarbital anesthesia.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes, daily
DETAILED CLINICAL OBSERVATIONS: No
BODY WEIGHT: Yes
FOOD CONSUMPTION: mean daily diet consumption calculated as g food/kg body weight/day weekly. Food consumption was not measured during the mating period.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
Oestrous cyclicity (parental animals):
Time of vaginal oestrus at mating.
Sperm parameters (parental animals):
No
Litter observations:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain day 1-4, physical or behavioural abnormalities
GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after 42 days of treatment were sacrificed after an 18 hours period of fasting by exsanguination under deep pentobarbital anesthesia
- Female animals: All surviving animals on day 4 of lactation, or on case of non-delivery on day 25 of pregnancy, or immediately after mating period in case of no-mating occured.

HISTOPATHOLOGY / ORGAN WEIGHTS
Males:
Organ weights: for all animals: epididymis, thymus, liver, kidney, testis.
Histopathological examination was performed for all animals on thymus, liver, kidneys, adrenal glands, bladder and testis. In the control group and the 750 mg dose group also heart, spleen, adrenal glands, bladder, testis and epididymis were examined.

Females:
In all animals ovaries and uterus were examined: Implantation sites in uterus were confirmed following Salwski colouring and Corpora lutea in ovaria were counted under a stereomicroscope. For all females expected of infertility orvaria were histopathologically examined.
Organ weights: all animals: thymus, kidney, liver.
Histopathological examination was performed for all animals on thymus, liver, kidneys, adrenal glands and bladder. In the control group and the 750 mg dose group also brain, heart, spleen, and uterus were examined.
Postmortem examinations (offspring):
Intra-abdominal organs were removed and fixed in 10% formalin solution and saved
Skeletal preparations were made for all pups necropsised on day 4 of the control and 750 mg dose groups and were examined for the presence of mutations and skeletal abnormalities.
Statistics:
Chi-quadrate test for conception and copulation rate. Otheres by Dunnett or Cheff’s method following evaluation for normal distribution by Barlett’s method. Otherwise, Kruskal-Wallis rank test was performed.
The level of significance was set to 5% and 1%.
Reproductive indices:
Mating performance:
Copulation index: (number of copulated pairs / number of mated pairs) x 100%
Fertility index: (number of pregnant animals/number of copulated animals) x 100%
Number of days before mating
Time of vaginal oestrus
Reproductive performance:
Number of pregnant females
Number of pregnant females with live pups
Gestation index: (Number of females bearing live pups / Number of pregnant females) x 100%
Gestation length
Number of corpora lutea
Number of implantation sites
Implantation index: (number of implantation sites/number of corpora lutea) x 100%
Number of pups born
Delivery index: (number of pups born/number of implantation sites) x 100%
Offspring viability indices:
Number of pups alive
Birth index: (number of pups alive on day 1/number of implantation sites) x 100%
Live birth index; (number of pups alive on day 1/number of pups born) x 100%
Sex ratio: (number of male pups alive on day 1 / number of female pups alive on day 1) x 100%
Viability index: (number of pups alive on day 4 / number of pups alive on day 1) x 100%
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Reproductive effects observed:
not specified

Mortality: No death was seen in males or females of any dose group.

Clinical bservations: Four male animals in 750 mg/kg dose group showed transient hematuria on the next or the second day after the initial administration. Hypersalivation in males and females was also seen from week 1 in 750 mg/kg dose group, week 2 in 300 mg/kg dose group and week 3 in 120 mg/kg dose group, and the number of symptomatic cases increased dose dependently.

Body weights: The males in 750 mg/kg dose group showed a significantly lower value (p<0.05, 0.01) consistently after week 1 of administration. Body weights for dose groups 300 mg/kg and less did not show significant difference compared to the control group.

Females: During pre-mating no significant difference was seen in any test substance dose group compared to the control group. During pregnancy, significantly low weight values (p<0.05, 0.01) were seen in 750 mg/kg dose group on day 7, 14, 20 of pregnancy. No significant difference was seen in dose groups 300 mg/kg and less compared to the control group.

After delivery, significantly low weights (p<0.05, 0.01) were observed on day 1 and 4 of nursing in 750 mg/kg dose group and on day 4 in 300 mg/kg dose group. No significant weight difference was seen in 120 mg/kg dose group compared to the control group.

Food consumption: Males: Except for a significantly lower food consumption seen during week 1 in 750 mg/kg dose group, no significant difference was seen in any test substance administration group compared to the control group.

Females: No significant difference was seen in any test substance administration group compared to the control group during pre-mating. During pregnancy: Significantly lower values (p<0.05, 0.01) were seen during day 0 – 7, 14 – 20 of pregnancy in 750 mg/kg dose group, during day 0 – 7, 7 – 14, 14 – 20 of pregnancy in 300 mg/kg dose group. After delivery: Although food consumption in 300 mg/kg dose group showed a significantly lower value (p<0.05), it was not a dose dependent change.

Hematology (Males): In dose groups 300 mg/kg or more, the number of white blood cells decreased dose dependently and significantly (p<0.01), and the ratio of lymphocytes tended to decrease. Furthermore, in 750 mg/kg dose group, the ratio of segmented neutrophils increased significantly (p<0.05). Although the number of red blood cells decreased significantly (p<0.05) in 300 mg/kg dose group, it was not a dose dependent change. No irregularity was found which was to be regarded as caused by test substance administration in hemoglobin concentration, hematocrit value, average volume of red blood cells, average amount of red blood cell hemoglobin, average red blood cell hemoglobin concentration, and number of thrombocytes.

Biochemistry (Males): Concentration of urine nitrogen was seen to increase dose dependently and significantly (p<0.05, 0.01) in dose groups 300 mg/kg or higher, and rather slight but significant (p<0.05, 0.01) increases were seen in GOT activation in dose groups 300 mg/kg or higher, GPT activation in 750 mg/kg dose group, and chlorine concentration in dose groups 300 mg/kg or higher. Also, in 750 mg/kg dose group, potassium concentration decreased rather slightly but significantly (p<0.01). Significant decreases (p<0.05, 0.01) were also seen in total protein concentration in 300 and 120 mg/kg dose groups, albumin concentration in 120 mg/kg dose group, inorganic phosphorus concentration in 300 and 120 mg/kg dose groups, calcium concentration in 300 mg/kg dose group. Although a-GTP activation showed a significant increase (p<0.01) in 120 mg/kg dose group, there was no dose dependency to this.

Autopsy:

Males: Livers in 6 samples in 750 mg/kg had dark colors. There was no other visual change that we surmised as caused by the test substance administration.

Females: Involution of thymus was seen in 8 cases of 750 mg/kg dose group (including 2 cases whose pups all died and 1 case of exclusion), 3 cases of 300 mg/kg dose group and 1 case of the control group. No other visual change which is surmised to have been caused by the test substance administration was observed, including 1 case in 120 mg/kg dose group which showed low formation of left corner of the uterus (non-pregnant case).

Organ Weight:

Males: As a result of measuring weights of thymus, liver, kidneys, testes, and epididymides, the ratios of kidneys and testes were significantly higher in 750 mg/kg dose group. As to the weights of other organs, there was no difference in the weight that we surmised as caused by the test substance administration except that the weight of thymus showed a tendency to decrease in 750 mg/kg dose group.

Females: (necropsy day 4 lactation): As a result of measuring weights of thymus, liver and kidneys, weight ratio of kidneys in dose groups higher than 300 mg/kg and weight ratio of liver in 750 mg/kg dose group showed a significantly higher value (p<0.01). No change that is surmised to have been caused by test substance administration was observed for other organs, except that thymus weight tended to decrease in dose groups higher than 300 mg/kg. (non-pregnant, cases in which all pups died, total embryo absorption, or excluded cases) Except that the thymus weights were remarkably low in two 750 mg/kg dose group cases in which all pups died, no change that is surmised to have been caused by the test substance administration.

Histopathology:

Males:

(Liver) Although fatty change in hepatic lobule area was seen in each group, change in test substance administration groups was smaller than the control group, and only 4 samples in 750 mg/kg dose group showed slight change. In addition, microgranuloma was also seen in each group, but there was no significant difference in frequency and degree from the control group. No other change was observed except for 1 case of foci of necrosis was seen in 750 mg/kg.

(Kidneys)

Although regenerated or atrophic tubule and eosinophilic body were seen in each group, there was no difference in frequency or degree from the control group. A case of dilated lumen of tubules in the control group, and a case of calculus in 300 mg/kg dose group were also seen. No change was seen except for 1 case of brown pigment deposition in each of 120 mg/kg dose group and the control group.

(Brain)

No change was seen in any group.

(Heart)

Although there were 2 cases of myocardial degeneration in 750 mg/kg dose group, and 2 cases of myocardial fibrosis in the control group, they were only slight changes.

(Spleen)

Brown pigment deposition and extramedullary hematopoiesis were seen in 750 mg/kg dose group and the control group, but there was no difference in both frequency and degree.

(Urinary Bladder)

A case of detached mucous epithelium was seen in each test substance dose group, and the cases from 750 and 120 mg/kg dose groups also showed hemorrhage and edema. Furthermore, thickening of mucous epithelium was seen in 11 cases in each of 750 and 300 mg/kg dose groups and 6 cases in 120 mg/kg dose group, and many cases were accompanied by cellular infiltration in lamina propria by lymphocytes or macrophages.

(Thymus)

There was no significant change except for 2 cases of involution were seen in 750 mg/kg dose group and 1 case of hemorrhage in each of 300 and 120 mg/kg dose groups and 2 cases of hemorrhage in the control group.

(Testes)

In 3 cases in each of 750 and 300 mg/kg dose groups and 5 cases in 120 mg/kg, seminiferous tubules with atrophy and decreased sperm cells were seen scatteredly.

(Epididymides)

Except that a foreign body giant cell was seen near spermatic granuloma in 1 case of the control group, no change was observed.

(Other Organs)

There was no change that we surmised as caused by test substance administration.

Females:

(Liver)

Foci of necrosis was seen in 1 case in each of 750 and 120 mg/kg dose groups, and the 750 mg/kg case also showed fatty change in the entire hepatic lobule. Furthermore, fatty change in the peripheral zone of hepatic lobule was seen in each group. However, the number of cases in 750 and 300 mg/kg dose groups was small. Microgranuloma was also seen in each group, but there was no difference in its frequency and degree.

(Kidneys)

A degenerated tubular epithelium, hemorrhage and collection of brown pigments in the lumen of tubules, and formation of vacuoles in the tubular epithelium were seen in 1 case of 750 mg/kg dose group (in which all pups died). A slight hemorrhage in the lumen of tubules was also seen in 1 case of 120 mg/kg dose group as well as a formation of vacuoles in the tubular epithelium in another case of 750 mg/kg (in which all pups died). Furthermore, 1 case of dilated lumen of tubule was seen in each of 120 mg/kg dose group and the control group, 1 to 4 cases of regenerated tubules in each group, and 1 case of calculus in the control group were also observed.

(Adrenals)

Increased vacuoles in zona fasciculata were seen in 2 cases of 750 mg/kg dose group (in which all pups died). In addition, deposition of brown pigments was seen in 2 cases of 750 mg/kg dose group and 1 case of 120 mg/kg dose group.

(Brain)

No change was observed in any group.

(Heart)

No change was observed in any group.

(Spleen)

Brown pigment deposition and extramedullary hematopoiesis were seen in 750 mg/kg dose group and the control group, but there was no difference in both frequency and degree.

(Urinary Bladder)

Thickening of mucous epithelium was seen in 7 cases of 750 mg/kg dose group and 12 cases of 300 mg/kg dose group, and 1 cases of 120 mg/kg dose group also showed a slight change. There was cellular infiltration in lamina propria of 2 to 4 cases from each test substance administration group.

(Thymus)

Involution was seen in each group including the control, but 5 cases from 750 mg/kg dose group and 4 cases from 300 mg/kg dose group showed strong involution.

(Uterus)

No change was observed except for dilated lumen was seen in 2 cases of 750 mg/kg dose group (one sterile case and another with total pup deaths).

(Ovaries)

No irregularity was observed in any case including sterile ones.

(Other Organs)

There was no change that we surmised as caused by test substance administration.

Mating Results: All pairs copulated, and there was no significant difference in conception rate between the control group and each of the test substance administration groups. Furthermore, the number of regressive oestrus since the pair was put to the same cage until the day of copulation confirmation did not show significant difference between the control group and each of the test substance administration groups.

Delivery and State of Nursing: Judging from the observation results on the first day of nursing in 750 mg/kg dose group, it was surmised that the state of delivery was unfavourable in 2 out of 10 samples (including 1 case of exclusion due to accidental water leak). All pups died by day 2 of nursing in these 2 cases. No other irregularity was seen in the delivery state and the nursing state of any administration group.

Number of Corpora Lutea, Implantations and Implantation Rate: There was no significant difference in the numbers of corpora lutea and implantations, and implantation rate between the control group and the test substance administration groups.

Birth Rate and Period of Pregnancy: There was no significant difference in the birth rate and the period of pregnancy between the control group and the test substance administration groups.

 

Offspring

Viability: Viability rate for the first day of nursing in 750 mg/kg dose group was significantly low (p<0.05). Delivery rate [(number of pups born / number of implantation sites)×100], birth rate [(number of pups born alive / number of implantation sites)×100], and viability rate for Day 4 of nursing did not show any significant difference between the control group and the test substance administration groups.

Body Weight: Body weights on day 1 of nursing (male + female) showed a small value (p<0.01) in 750 mg/kg dose group, and if seen by sex, value for the males showed a significant difference (p<0.05) compared to the control group. There was no significant difference in the pup weight on day 4 of nursing between the control group and the test substance administration groups.

Malformations: No outer and internal irregularity which is surmised to have been caused by the test substance administration was seen in visual observation on the day of birth and autopsy of dead pups and of all born pups on the day 4 of nursing. In skeletal observation performed on the 750 mg/kg dose group and the control group, 1 case of cleft sternum among the 6 pups which died, and another case of composite deformation in cranial bone, spine and ribs were seen in 750 mg/kg dose group, but there was no significant difference in frequency between the two groups.

Conclusions:
NOEL for reproduction: 300 mg/kg bw/day. These results were used for read-across to Tosylchloramide sodium.
Executive summary:

OECD 422, Combined Repeat Dose and Reproductive/Developmental Toxicity Screening Test in rat by gavage under GLP. Dose levels: 0 (vehicle, 5% Arabic gum), 120, 300, 750 mg/kg/day, with 13 animals per dose group per sex. Dosing period: Females, from 14 days before mating to day 3 of lactation. Males 42 days.

 Mating performance and fertility were not affected by the test compound. Two of 10 females in the high-dose group showed signs of difficult delivery and all of their offspring died by day 3 of lactation. Reproduction parameters (i.e., duration of gestation, numbers of corpora lutea, implantations and resorptions, litter size, and sex distribution) were comparable among all four groups, including the control. However, significant decreases in both survival rate and body weight of newborns on day 0 of lactation were observed in the high-dose group. Morphological observations of offspring revealed no teratogenic effects of 4-methylbenzenesulfonamide.

NOEL for P generation: 300 mg/kg/day.

NOEL for F1 generation: 300 mg/kg/day.

These results were used for read-across to Tosylchloramide Sodium.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
165 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
There are two studies available (OECD 422 screening study, and a two-generation study (OECD 416), both of high quality.
The two-generation study (OECD416 with p-TSA is selected as the most relevant for the evaluation of reproduction toxicity. The selected NOAEL represents the lowest reproduction/development NOAEL of 3000 ppm (corresponding to 165-237 mg/kg bw/day in males and 232 -499 mg/kg bw/day in females).
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

There are two studies available for evaluation of reproduction toxicity:

70-55-3, Toxicity to reproduction 2-gen oral-rat with p-TSA, NOTOX 474064, 2007 A full two-generation study in rats has been performed under GLP and according to OECD 416 with p-TSA administration by dietary admixture at levels of 0, 1000, 3000 and 10,000 ppm. All animals were subjected to daily clinical observation. Body weight and food consumption were measured over the treatment period. The regularity and duration of the estrous cycle was examined. At necropsy, macroscopic observations and organ weights were recorded. Sperm morphology, motility and count were assessed. A histopathological examination was performed on all reproduction organs and tissues. Reproduction parameters, breeding data and pup development were assessed. No effects were observed at 1000 ppm. At 3000 ppm terminal body weights of F0 and F1 parent animals at necropsy were decreased which resulted in relative organ weight changes for the brain and kidneys. At 10,000 ppm the terminal body weights at necropsy were decreased which resulted in relative organ weight changes for the brain, liver, kidneys, seminal vesicles, adrenals, and spleen. In the F1 generation also testes, prostate, and pituitary gland showed relative organ weight changes. Also lower body weights for male and female pups were observed which resulted in organ weight changes for the brain, spleen and thymus and a delay in vaginal opening and balano-preputial separation in F1 animals. Based on these findings thestudy concluded upon:

NOAEL parental of 1000 ppm (males 52 -78 (average 65) mg/kg bw/day; females 75-161 mg/kg bw/day).

NOAEL reproduction and breeding of at least 10000 ppm (males 566 -832 mg/kg bw/day; females 733 -1631 mg/kg bw/day)

NOAEL developmental toxicity of 3000 ppm (males 165-237 mg/kg bw/day; females 232 -499 mg/kg bw/day)

70-55-3, Toxicity to reproduction OECDTG 422, oral-rat with p-TSA, MHW Japan, 1994 OECD 422, Combined Repeat Dose and Reproductive/Developmental Toxicity Screening Test in rat by gavage under GLP. Dose levels: 0 (vehicle, 5% Arabic gum), 120, 300, 750 mg/kg/day, with 13 animals per dose group per sex. Dosing period: Females, from 14 days before mating to day 3 of lactation. Males 42 days. Mating performance and fertility were not affected by the test compound. Two of 10 females in the high-dose group showed signs of difficult delivery and all of their offspring died by day 3 of lactation. Reproduction parameters (i.e., duration of gestation, numbers of corpora lutea, implantations and resorptions, litter size, and sex distribution) were comparable among all four groups, including the control. However, significant decreases in both survival rate and body weight of newborns on day 0 of lactation were observed in the high-dose group. Morphological observations of offspring revealed no teratogenic effects of 4-methylbenzenesulfonamide.

NOEL for P generation: 300 mg/kg/day

NOEL for F1 generation: 300 mg/kg/day

Both available studies do not indicate a specific concern regarding reproduction toxicity. Reproductive effects that have been observed at highest dose levels can bet attributed to the lower parental condition as indicated by the lower body weight, and subsequent lower body weight of the pups. This in turn can affect development in pups as indicated by observeddelay in vaginal opening and balano-preputial separation. No effects were seen on fertility and breeding results.

The LOAEL from the two-generation study is at 10,000 ppm (males 566 -832 mg/kg bw/day; females 733 -1631 mg/kg bw/day), which compares well with the reproduction LOAEL of 750 mg/kg bw/day by gavage from the OECD 422 study.

The overall NOAEL for reproduction toxicity is set at 3000 ppm (males 165-237 mg/kg bw/day; females 232 -499 mg/kg bw/day), a level that correlates to the level at which parental toxicity becomes visible. This level is also at the same order of magnitude as the NOAEL of 300 mg/kg bw/day observed in the OECD 422 study following dosing by gavage.

These results were used for read-across to Tosylchloramide Sodium.

Effects on developmental toxicity

Description of key information

Developmental toxicity (OECDTG 414) with rabbits: NOAEL 50 mg/kg bw/day in males and 953 mg/kg bw/day in females. (read across from o/p-TSA)

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
30 April 1984 - 7 March 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Range finding study for study IR-84-238. Therefore only a summary of the study is provided.
GLP compliance:
yes
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
1 500 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Based on these results, dose levels of 50, 250 and 500 mg/kg/day were selected for a definitive teratology study in rats with Sancticizer 9. These results were used for read-across to Tosylchloramide sodium.
Executive summary:

Mated Charles River COBS CD rats consecutively assigned to one control and five treatment groups of five animals each were used in this range-finding study to detewine dosage levels of Santicizer 9 for a teratology study. Dosage levels of 100, 500, 1000, 1500, and 2000 mg/kg/day were administered orally by gavage as a single daily dose on days 6 through 15 of gestation at a constant volume of 10 ml/kg. The control group received the vehicle only, corn oil, on a comparable regisen. Uterine examinations were performed on all surviving fenales on gestation day 20.

Maternal toxicity as evidenced by deaths, prescheduled sacrifices, decreased or loss of activity following dosing and inadequate grooning was noted in the 1000, 1500 and 2000 mg/kg/day groups. Postdose inactivity was also noted in one 500 mg/kg/day female exclusively on the first day of treatment. Body weight change values for the 500, 1000, 1500 and 2000 mg/kg/day groups during the treatment period (days 6 to 15) indicated reduced gains or losses in body weight in relation to the control value. A dose related trend concerning reduced gain during the overall gestation period (days 0 to 20) was noted in all treated groups when compared to that of the control.

Embryolethality, as indicated by an increase in postimplantation losses/dam with a concurrent decrease in viable fetuses/dam was noted in the 1500 and 2000 mg/kg/day groups when compared to the control. Whole litter resorption occurred in one feamles in each of these groups. The uterine examination parameter values of the females in the 100, 500 and 1000 mg/kg/day group were not meaningfully different from those of the control group.

Based on these results, dose levels of 50, 250 and 500 mg/kg/day were selected for a definitive teratology study in rats with Sancticizer 9.

These results were used for read-across to Tosylchloramide sodium.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 July 1984 - 15 August 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was performed under GLP and according to a method similar to currently internationally accepted guidelines. No verification of dose levels, and no information on food consumption. Exposure from implantation (day 6) through day 15 of gestation instead of from implantation until one day prior to the day of scheduled kill (day 19). Gravid uterus weight was not determined.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
Performed in accordance with Series 83-3 of the Environmental Protection Agency Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation: Human and Domestic Animals, issued November, 1982.
Devations:
- Gravid uterus weight was not determined.
- No verification of dose levels
- No information on food consumption.
- Exposure from implantation (day 6) through day 15 of gestation instead of from implantation until one day prior to the day of scheduled kill (day 19).
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: COBS CD
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, Michigan, USA
- Age at study initiation: 70 days
- Weight at study initiation: The rats were 118 days old at the time of mating and weighed between 255 and 346g on gestation day 0.
- Fasting period before study: Not applicable
- Housing: The rats were housed individually in suspended wire-mesh cages from receipt until sacrifice except during mating when one male and one female were placed together.
- Diet (e.g. ad libitum): ad libitum, basal Laboratory diet of Purina Certified Rodent Chow #5002
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: 48 day acclimation period


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2 ± 0.35
- Humidity (%): 67 ± 13.4
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 18 July 1984 - 15 August 1985
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The appropriate amount of Santicizer 9 for each group was ground daily with a mortar and pestle, weighed, and suspended i n the vehicle, Mazola corn oil, using a tissue homogenizer. The test material suspension was then transferred to a graduated mixing cylinder and additional vehicle added to yield a sufficient volume of prepared test material.
The suspension was shaken by hand and dispensed into a capped container. Prior to administration, the suspension was stirred using a magnetic stit
bar and stir plate.

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: The test article was prepared at concentrations to permit the administration of dosage levels of 50, 250 and 500 mg/kg/dap at a dosage volume of 10 ml/kg. Individual dosages were determined from individual body weights recorded on gestation days 6, 9 and 12.
- Amount of vehicle (if gavage): max. 10 ml/kg.
- Lot/batch no. (if required): no data
- Purity: no data
Analytical verification of doses or concentrations:
no
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: no data
- After "no data" days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no data
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- Any other deviations from standard protocol: none
Duration of treatment / exposure:
single daily dose on days 6 through 15 of gestation
Frequency of treatment:
Single daily dose
Duration of test:
Days 6 through 15 of gestation
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: range finding study. For summary see "1333-07-9, Developmental toxicity / teratogenicity, Leng, 1985, RS"
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Prior to treatment, the females were observed twice daily for mortality and overt changes in appearance and behavior. They were
observed twice daily for mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily on days 6 through 15 of gestation.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual maternal body weights were recorded on gestation days 0, 6, 9, 12, 16 and 20.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: The abdominal and thoracic cavities and organs of the dams wesere examined for grossly evident morphological changes and the carcasses discarded. Maternal tissues vere preserved in 10% neutral buffered formalin for possible histopathological examination as deemed necessary by the gross findings.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: The number and location of viable and nonviable fetuses, uteri from females that appeared nongravid uere opened and placed in 10% anmonium sulfide solution for detection of implantations.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litte
- Head examinations: No: included in soft tissue examination, only applicable for non-rodents
Statistics:
All statistical analyses compared the treatment groups with the control group, with the level of significance at P<0.05 and p<0.01. All means were accompanied by standard deviations. Male to female fetal sex ratios and the proportions of litters with malformations were compared using the Chi-square test criterion with Yates' correction for 2 x 2 contingency tables and/or Fisher's exact probability test as described by siegel to judge significance of differences.
The proportions of resorbed and dead fetuses and postimplantation losses were compared by the Mann-Whitney U-test as described by Siegel and Weil to judge significance of differences.
Mean numbers of corpora lutea, total implantations, live fetuses, mean fetal body weights, and mean maternal body weights and body weight changes were compared by analysis of variance (one-way classification). Barelett's test for homogeneity of variances and the appropriate t-test (for equal or unequal variances) as described by Steel and Torrie using Dunnet t 's multiple comparison tables to judge significance of differences.
Historical control data:
Included in the study.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Survival was 100% among the control, 50, 250 and 500 mg/kg/day groups. The test article did not produce meaningful differences in maternal appearance and behavior at any dosage level. There was no test item related effect on pregnancy rate.
Clinical signs observed in some groups inclusive of the control, included the following : alopecia .and scabbing, sparse haircoat,
and malligned upper incisors with associated ocular discharge.
The following gross changes were noted during necropsy in the treated and control groups: hydronephrosis with or without distended ureters, misshapen kidney, distended uterus and cervix, and adherence of the liver to the kidney.
The test article had a marked inhibitory effect on maternal body weight gain at 250 and 500 mg/kg/day during the overall treatment interval.Moreover, marked reductions in gain were evident at 500 mg/kg/day during the overall gestation interval. Weight loss was observed during the gestation day 6 to 9 subinterval for both groups. All of these decreases were statistically significant. No similar effect was observed at 50 mg/kg/day.
An increase in postimplantation loss was noted for the 250 and 500 mg/kg/day groups, relative to the control. The increase was statistically significant at 500 mg/kg/day. The test article had no effect on the i.e., corpora lutea, total implantations, viable litter size, and sex Distribution, at 500 mg/kg/day and less.
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
A treatment-related trend of fetal body weight inhibition was observed at all dosage levels. Fetal body weights were statistically significantly less than the control at 250 and 500 mg/kg/day. Weight inhibition, although very slight and not statistically significant, was also observed at 50 mg/kg/day and was deemed compound related as it was considered part of the trend observed at higher dosage levels.
The incidence of fetal malformations occurring in the treated groups was not meaningfully different from the control. A single instance each of a tail anomaly with anal atresia, and a vertebral anomaly occurred in a 250 mg/kg/day litter. Also, one 500 mg/kg/day fetus exhibited a retroesophageal aortic arch. No malformations occurred in the control and 50 mg/kg/day animals.
With respect to developmental variations, the number of litters exhibiting unossification of sternebrae #5 and/or #6 was increased exclusively at 500 mg/kg/day relative to the control. The remaining variations were noted in the control and treated groups at similar incidence or occurred as an isolated incidence and were deemed biologically irrelevant.
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: developmental toxicity
Remarks on result:
other: Significant effect in the fetuses were only observed at dose level where maternal toxicty was also observed.
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Based on these results, o/p-TSA did not produce a teratogenic effect when administered orally to mated Charles River COBS CD rats at 500 mg/kg/day and less. These results were used for read-across to Tosylchloramide sodium.
Executive summary:

A study was performed under GLP and according to a method similar to currently internationally accepted guidelines. Performed in accordance with Series 83-3 of the Environmental Protection Agency Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation: Human and Domestic Animals, issued November, 1982. Mated Charles River COBS CD rats consecutively assigned to one control and three treatment groups of 25 animals each were used to determine the teratogenic potential of Santicizer 9.

Dosage levels of 50, 250 and 500 mg/kg/day were administered orally by gavage as a single daily dose on days 6 through 15 of gestation at a constant volume of 10 ml/kg. The control group received the vehicle only, corn oil, on a comparable regimen. Cesarean sections were performed on all surviving females on gestation day 20 and the fetuses were removed for teratologic evaluation.

Survival was 100% among all groups on study. Santicizer 9 produced meaningful changes in maternal body weight gain and several Cesarean section parameters when administered at 250 and 500 mg/kg/day. Maternal weight loss was evident during a portion of the treatment interval at 250 and 500 mg/kg/day and both groups exhibited significant (p<0.01) reductions in weight gain during the overall treatment period. Statistically significant reductions were also noted at 500 mg/kg/day during the overall gestation period.

Fetotoxicity was apparent at 250 and 500 mg/kg/day as evidenced by increased postimplantation loss which was statistically significant at 500 mg/kg/day. In addition, a treatment-related trend of fetal body weight inhibition was noted at all dosage levels and values were significantly different from the control, p<0.01 at 250 and 500 mg/kg/day. An increase in the number of litters exhibiting unossification of sternebrae #5 and/or #6 was noted exclusively at 500 mg/kg/day and was correlated with fetal body weight reduction. Maternal appearance and behavior and the incidence of fetal malformations were not affected at any dosage level.

Based on these results, Santicizer 9 did not produce a teratogenic effect when administered orally to mated Charles River COBS CD rats at 500 mg/kg/day and less.

These results were used for read-across to Tosylchloramide sodium.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007, March 14 - July 27
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study performed according to OECD and EU guidelines and according to GLP principles. In principle an evaluation of two separately treated cohorts.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
See below
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
See below
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
See below
Principles of method if other than guideline:
Protocol deviations:
1 On 05 February 2007 (Dose range finding study, NOTOX Project 474086, see Appendix 5) and on 24 March 2007 (Main study), no water consumption was determined. Evaluation: Sufficient data available for evaluation of effects on water consumption.
2 No skeletal examinations were performed on fetuses 03 and 04 from animal 31. Thorough examination was not possible as skeletal preparations were fallen into individual bones, possibly due to the use of an incorrect fixative. Evaluation: The examination that was performed did not reveal findings related to treatment. There were sufficient fetuses left in the litter for evaluation.
3 On 17 and 23 July 2007 animals have been observed, however observations have not been entered into the computer. On both days there were no remarkable changes when compared to the observation on the day before. Evaluation: Animals were observed, only no on-line data is available.
4 Temporary deviations from the minimum and maximum level of temperature occurred. Evaluation: Laboratory historical data do not indicate an effect of the deviations.
The study integrity was not adversely affected by the deviations.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan France SARL, Gannat, France
- Age at study initiation: Part I: Females were approximately 18-20 weeks. Part II: Females were approximately 21-23 weeks.
- Weight at study initiation: Day 0 post coitum: 2478-3887 g
- Fasting period before study: Not applicable
- Housing: Females were individually housed in labelled cages with perforated floors
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: Part I: At least 13 days prior to insemination. Part II: At least 3 weeks prior to insemination.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16.9-24.1°C. Temporary deviations from the minimum and maximum level of temperature occurred. Laboratory historical data do not indicate an effect of the deviations.
- Humidity (%): 31 - 81%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was mixed without the use of a vehicle, directly with some powder feed (premix) and subsequently mixed with the bulk of the diet. Milli-U water (approximately 12% in total) was added to aid pelleting. The pellets were dried for approximately 24 hours at 35°C before storage.

DIET PREPARATION
- Rate of preparation of diet (frequency): Diets were prepared at least once every two weeks.
- Mixing appropriate amounts with Standard powder rabbit diet
- Storage temperature of food: room temperature
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were analyzed using 30 and 60 minutes ultrasonication in Part I and using 60 minutes ultrasonication in Part II. The concentrations analysed in diets of Group 2, Group 3 and Group 4 were between 80 and 108% (Part I) using 30 minutes of ultrasonication and between 86% and 114% (Part I) and 81% to 106% (Part II) of target using 60 minutes of ultrasonication for extraction. For studies using diet, a range of 80-120% is considered acceptable.
Diets are considered to be homogeneous with a coefficient variation <10%.
Details on mating procedure:
Not applicable. Animals are inseminated.
Duration of treatment / exposure:
Day 7 - 29 post coitum.
Frequency of treatment:
Ad libitum
Duration of test:
30 days
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
equivalent to 0 ppm
Dose / conc.:
41 mg/kg bw/day (actual dose received)
Remarks:
equivalent to 1000 ppm
Dose / conc.:
113 mg/kg bw/day (actual dose received)
Remarks:
equivalent to 3000 ppm
Dose / conc.:
367 mg/kg bw/day (actual dose received)
Remarks:
equivalent to 11000 ppm
No. of animals per sex per dose:
27 at 0 and 3000 ppm (24 in part I, 3 in part II)
30 at 1000 and 11000 ppm (24 in part I, 6 in part II)
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Based on a range finding study
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes : mortality
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from Day 0 post-coitum onwards.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 4, 7, 10, 13, 16, 20, 23, 26 and 29 post-coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Days 0-4, 4-7, 7-10, 10-13, 13-16, 16-20, 20-23, 23-26 and 26-29 post-coitum.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Daily from Day 0 post-coitum onwards.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Day 29 post-coitum
- Organs examined: External, thoracic and abdominal examinations, ovary and uterine horn, female genital tract including the placentas
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- The number of corpora lutea (ovaries in situ)
- The weight of the gravid uterus
- The number and distribution of live and dead foetuses
- The number and distribution of embryo-foetal deaths
- The weight of each foetus
- The sex of each foetus (during further foetal examination)
- Externally visible macroscopic foetal abnormalities.
Fetal examinations:
- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [all per litter ]
- Skeletal examinations: Yes: [all per litter ]
- Head examinations: Yes: [one-half of the foetuses per litter ]
Statistics:
The following statistical methods were used to analyse the data:
• If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex (Dunett, 19955).
• The Steel-test (many-to-one rank test) was applied instead of the Dunnett-test if the data could not assumed to follow a normal distribution (Miller, 1981).
• The Fisher-exact test was applied to frequency data (Fisher, 1950).
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Indices:
Pre-implantation loss: (Number of corpora lutea - number of implantation sites) x 100 /Number of corpora lutea
Post-implantation loss: (Number of implantation sites - number of live foetuses) x 100 /Number of implantation sites


Historical control data:
Historical control data on the background incidence of foetal malformations and developmental variations in New Zealand White rabbits from the same source was used for comparison with concurrent controls in the study.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
At the high dose group (11000 ppm), decreased body weight and body weight gain were noted, which were most pronounced on the first days of treatment. Although an increase in body weight and body weight gain was noted from Day 10 post-coitum onwards, body weight remained lower for the entire treatment period. Body weight gain at 11000 ppm was also reduced at necropsy after correction for uterus weight. In addition, reduced food consumption was noted mainly during the first days of treatment (Days 7-10 post-coitum). Food consumption improved from Day 10 post-coitum onwards and had returned to control values at the end of treatment. The findings in body weight and food consumption were accompanied by a reduction in water consumption from Day 7 post-coitum onwards, which increased to normal values at the end of treatment.

At the intermediate dose group (3000 ppm), similar effects on body weight, food- and water consumption were noted during the first days of treatment. However, these effects were less severe and recovered sooner and were therefore considered to be transient.

No maternal toxicity was observed at the low dose group (1000 ppm).
Key result
Dose descriptor:
NOAEL
Effect level:
113 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
3 000 ppm
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
No effects on pre- and post implantation loss, litter size and sex ratio were noted.
A low pregnancy rate was observed during Part I of the study. As treatment started after implantation and as the low pregnancy rate was observed in all groups, including the control group, in absence of a dose response relationship, the low pregnancy was not treatment related. The low pregnancy rate might be due to stress caused by periods of loud noises due to on-site construction work.
In both Parts I and II and after combining data of these parts, a trend towards reduced body weight of fetuses (statistically not significant) was noted at the high dose group (11000 ppm). This was considered to be related to the decreased maternal body weight at 11000 ppm.
The total number of malformations observed in fetuses (litters) in the different treatment groups were 3(3), 0(0), 4(3) and 11(8) in the control, 1000, 3000 and 11000 ppm groups, respectively. The only treatment related malformations were vertebral anomalies with or without associated rib anomaly, which were noted in 4 fetuses from four different litters in Part I. After combining data of both Part I and II, a statistically significant increase in the mean litter proportion of fetuses with vertebral anomaly with or without associated rib anomaly, a skeletal malformation, was noted at a dose level of 11000 ppm, whilst this finding was not observed for fetuses in the control, 1000 and 3000 ppm groups. In addition, no such findings were observed in Part II of the study.
Vertebral anomalies with or without associated rib anomaly had not been seen in the concurrent control group. Historical control data revealed that these anomalies had occurred at a fetal incidence of 0.4% in control animals (range 0.0% – 1.2%).
To further determine the toxicological significance of vertebral anomalies with or without associated rib anomaly, all fetuses of the dose range finding study were skeletally examined. Results of skeletal evaluations did not show vertebral anomalies with or without associated rib anomaly up to a dose level of 20000 ppm.
Based on the findings in this current study, a direct fetal effect of the test substance at 11000ppm in the diet to cause vertebral anomalies with or without associated rib anomaly can not be ruled out. However it might also be related to increased maternal stress in these test substance treated groups, since it was not observed in any of the Part II animals in this study, or in animals dosed with up to 20000 ppm in the dose ranging study.
Key result
Dose descriptor:
NOAEL
Effect level:
113 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEL
Effect level:
3 000 ppm
Sex:
male/female
Basis for effect level:
other: developmental toxicity
Remarks on result:
other: - Statistically significant increase in the mean litter proportion of vertebral anomalies with or without associated rib anomaly at 11000 ppm.
Abnormalities:
not specified
Developmental effects observed:
not specified

The table below shows the incidence of vertebral anomalies with or without associated rib anomaly in the main and dose range findings study, when compared to the total number of fetuses and litters (as well as total malformations and developmental delay):

Part I

Fetuses (Litter) with Finding

Total # Fetuses

Total # Litters

A

B

C

Control

1(1)

0(0)

0(0)

93

17

1000 ppm

0(0)

0(0)

0(0)

96

15

3000 ppm

3(2)

0(0)

2(1)

104

17

11000 ppm

11(8)

4(4)

3(3)

88

14

Part II and Ranger Combined

Fetuses (Litter) with Finding

Total # Fetuses

Total # Litters

A

B

C

Control

2(2)

0(0)

0(0)

52

8

1000 ppm

0(0)

0(0)

1(1)

14

3

3000 ppm

1(1)

0(0)

0(0)

17

3

5000 ppm

2(2)

0(0)

0(0)

44

6

10000/11000 ppm

1(1)

0(0)

0(0)

66

12

20000 ppm

0(0)

0(0)

0(0)

32

4

 

A = Total malformations

B = Malformations: vertebral anomalies with or without associated rib anomaly

C = Developmental delay: Vertebral centra anomalies

Conclusions:
Rabbits dosed with 11000 ppm p-TSA showed evidence of toxicity with reduced body weight which remained lower than controls for the entire treatment period. Body weight gain at 11000 ppm was also reduced at necropsy after correction for uterus weight. Transient effects on body weight were noted at 3000 ppm. No treatment related toxicity was observed at 1000 ppm.

Based on the results obtained in this prenatal developmental toxicity study, it is concluded that dietary administration of p-TSA to pregnant rabbits during the period of organogenesis at a high dose level of 11000 ppm was associated with a statistically significant increase in the mean litter proportion of vertebral anomalies with or without associated rib anomaly. However, whether this was related to p-TSA or to a combination of p-TSA with incidental stress which occurred during the first part of the study could not be determined, since the findings were not observed in any of the Part II animals in this study, or in animals dosed up to 20000 ppm in the dose ranging study. No test substance related findings on fetal morphology occurred at dose levels of 1000 and 3000 ppm. Therefore, a dosage level of 3000 ppm (113 mg/kg body weight/day) was considered to be the No Observed Adverse Effect Level (NOAEL) for embryo/fetal developmental toxicity.

Furthermore, based on the effects on body weight, food and water consumption, the maternal No Observed Adverse Effect Level (NOAEL) for p-TSA was established as being 3000 ppm (113 mg/kg body weight/day).

These results were used for read-across to Tosylchloramide sodium.
Executive summary:

STUDY OUTLINE

Four groups of 27 to 30 New Zealand White rabbits were inseminated (Day 0 post-coitum) and exposed by dietary exposure to 0, 1000, 3000 and 11000 ppm (equivalent to 0, 41, 113 and 367 mg/kg body weight/day respectively) from Days 7 to 29 post-coitum.

The study consists of Part I and II. In Part I of the study 24 animals/group were inseminated. A low pregnancy rate was observed in all groups, which resulted in an insufficient number of litters; seventeen litters in the control and mid dose group, fifteen litters in the low dose group and fourteen litters in the high dose group. Therefore, 18 additional animals (3 animals/group in Groups 1 and 3, and 6 animals/group in Groups 2 and 4) were added to the study (Part II), which resulted in the addition of three litters in the control, low and mid dose group and six litters in the high dose group. In Part I and II, treatment and study procedures were comparable.

EVALUATED PARAMETERS

Females were checked daily for the presence of clinical signs. Body weight and food consumption of females was determined at periodic intervals; and water consumption daily. Diet analysis was performed on prepared diets.

All animals surviving to Day 29 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. The ovaries and uterine horns were dissected and examined for the number of corpora lutea, the weight of the gravid uterus, the number and distribution of live/dead fetuses and embryo-fetal deaths, the weight of each fetus, fetal sex and externally visible fetal macroscopic abnormalities. All live fetuses were euthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin’ fixative and subsequently sliced, all fetuses were dissected and examined for visceral anomalies and subsequently fixed in 96% aqueous alcohol and stained with Alizarin Red S for skeletal examinations.

RESULTS

Accuracy, homogeneity and stability of diet preparations were demonstrated by analyses.

Maternal findings

At the high dose group (11000 ppm), decreased body weight and body weight gain were noted, which were most pronounced on the first days of treatment. Although an increase in body weight and body weight gain was noted from Day 10 post-coitum onwards, body weight remained lower for the entire treatment period. Body weight gain at 11000 ppm was also reduced at necropsy after correction for uterus weight. In addition, reduced food consumption was noted mainly during the first days of treatment (Days 7-10 post-coitum). Food consumption improved from Day 10 post-coitumonwards and had returned to control values at the end of treatment. The findings in body weight and food consumption were accompanied by a reduction in water consumption from Day 7 post-coitum onwards, which increased to normal values at the end of treatment.

At the intermediate dose group (3000 ppm), similar effects on body weight, food- and water consumption were noted during the first days of treatment. However, these effects were less severe and recovered sooner and were therefore considered to be transient.

No maternal toxicity was observed at the low dose group (1000 ppm).


Developmental findings

No effects were noted on pre- and post implantation loss, litter size and sex ratio.

A low pregnancy rate was observed during Part I of the study. As treatment started after implantation and as the low pregnancy rate was observed in all groups including the control group in absence of a dose response relationship, the low pregnancy was not treatment related. The low pregnancy rate might be due to stress caused by periods of loud noises due to construction work.

In both Parts I and II and after combining data of these parts, a trend towards reduced body weight of fetuses (statistically not significant) was noted at the high dose group (11000 ppm). This was considered to be related to the decreased maternal body weight at 11000 ppm.

The total number of malformations observed in fetuses (litters) in the different treatment groups were 3(3), 0(0), 4(3) and 11(8) in the control, 1000, 3000 and 11000 ppm groups, respectively. The only treatment related malformations were vertebral anomalies with or without associated rib anomaly, which were noted in 4 fetuses from four different litters in Part I. After combining data of both Part I and II, a statistically significant increase in the mean litter proportion of fetuses with vertebral anomaly with or without associated rib anomaly, a skeletal malformation, was noted at a dose level of 11000 ppm, whilst this finding was not observed for fetuses in the control, 1000 and 3000 ppm groups. In addition, no such findings were observed in Part II of the study.

Vertebral anomalies with or without associated rib anomaly had not been seen in the concurrent control group. Historical control data revealed that these anomalies had occurred at a fetal incidence of 0.4% in control animals (range 0.0% – 1.2%).

To further determine the toxicological significance of vertebral anomalies with or without associated rib anomaly, all fetuses of the dose range finding study were skeletally examined (NOTOX Project 474086, Appendix 7C). Results of skeletal evaluations did not show vertebral anomalies with or without associated rib anomaly up to a dose level of 20000 ppm.

The table below shows the incidence of vertebral anomalies with or without associated rib anomaly in the main and dose range findings study, when compared to the total number of fetuses and litters:

Part I:

finding

fetuses (litters)

Total number of fetuses

Total number of litters

Control:

0 (0)

93

17

1000 ppm

0 (0)

96

15

3000 ppm

0 (0)

104

17

11000 ppm

4 (4)

88

14

Part II and range finding combined:

finding

fetuses (litters)

Total number of fetuses

Total number of litters

Control:

0 (0)

52

8

1000 ppm

0 (0)

14

3

3000 ppm

0 (0)

17

3

5000 ppm

0 (0)

44

6

10000/11000 ppm

0 (0)

66

12

20000 ppm

0 (0)

32

4

Based on the findings in this current study, a direct fetal effect of the test substance at 11000 ppm in the diet to cause vertebral anomalies with or without associated rib anomaly can not be ruled out. However it might also be related to increased maternal stress in these test substance treated groups, since it was not observed in any of the Part II animals in this study, or in animals dosed with up to 20000 ppm in the dose ranging study.

CONCLUSION

Rabbits dosed with 11000 ppm p-TSA showed evidence of toxicity with reduced body weight which remained lower than controls for the entire treatment period. Body weight gain at 11000 ppm was also reduced at necropsy after correction for uterus weight. Transient effects on body weight were noted at 3000 ppm. No treatment related toxicity was observed at 1000 ppm.

Based on the results obtained in this prenatal developmental toxicity study, it is concluded that dietary administration of p-TSA to pregnant rabbits during the period of organogenesis at a high dose level of 11000 ppm was associated with a statistically significant increase in the mean litter proportion of vertebral anomalies with or without associated rib anomaly. However, whether this was related to p-TSA or to a combination of p-TSA with incidental stress which occurred during the first part of the study could not be determined, since the findings were not observed in any of the Part II animals in this study, or in animals dosed up to 20000 ppm in the dose ranging study. No test substance related findings on fetal morphology occurred at dose levels of 1000 and 3000 ppm. Therefore, a dosage level of 3000 ppm (113 mg/kg body weight/day) was considered to be the No Observed Adverse Effect Level (NOAEL) for embryo/fetal developmental toxicity.

Furthermore, based on the effects on body weight, food and water consumption, the maternal No Observed Adverse Effect Level (NOAEL) for p-TSA was established as being 3000 ppm (113 mg/kg body weight/day).

These results were used for read-across to Tosylchloramide sodium.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
High. There is one recent GLP full OECD 414 comparable study available in rats showing no teratogenic effects at toxic dose levels of TSA (o,p -Toluene sulphonamide) and additionally one recent GLP full OECD 414 study available in rabbits, and information from an OECD 422 screening study in rat which included evaluations for skeletal malformations.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

1333 -07 -9, Developmental toxicity / teratogenicity (OECD414, oral-rat with o/p-TSA), Int. R&D Corp., 401 -290, 1985

There is one GLP compliant study on o/p-TSA available that is comparable to an OECD 414 study available in rats.

o/p-TSA was administered orally by gavage to groups of 25 mated rats per dose level of 0 (vehicle only, corn oil), 50, 250 and 500 mg/kg/day on days 6 through 15 of gestation. Cesarean sections were performed on all surviving females on gestation day 20 and the foetuses were removed for teratologic evaluation.

Survival was 100% among all groups on study. At 250 and 500 mg effects were observed in maternal body weight gain and several Cesarean section parameters. Statistically significant reductions were also noted at 500 mg/kg/day during the overall gestation period. Foetotoxicity was apparent at 250 and 500 mg/kg/day as evidenced by increased postimplantation loss which was statistically significant at 500 mg/kg/day. In addition, a treatment-related trend of foetal body weight inhibition was noted at all dosage levels and values were significantly different from the control, p<0.01 at 250 and 500 mg/kg/day. An increase in the number of litters exhibiting delayed ossification was noted exclusively at 500 mg/kg/day and was correlated with foetal body weight reduction. Maternal appearance and behaviour and the incidence of foetal malformations were not affected at any dosage level. Based on these results, o/p-TSA did not produce a teratogenic effect when administered orally to mated rats at 500 mg/kg/day and less.

A NOAEL of 50 mg/kg bw/d was established for maternal and developmental toxicity.

Cross-reading (RA) between Toluenesulphonamides (o-TSA, p-TSA and o/p-TSA) is justified and already previously described by Houthoff, E. andGyimesi, M. (2013) and was submitted to ECHA in the dossier of o/p-TSA and this dossier. The available data confirm the comparable hazard profiles for p-TSA and o-TSA covering all physical-chemical ecotoxicological en toxicological endpoints. Further support is provided by the available data on o/p-TSA. As this is basically a mixture of p-TSA and o-TSA its study results can be expected to reflect the same hazard profile.

70-55-3, Developmental toxicity / teratogenicity OECD 414, oral-rabbit with p-TSA, Notox, 474097, 2008

p-TSA was evaluated in a Prenatal Developmental Toxicity Study following OECD 414 guidelines under GLP. Four groups of twenty-four New Zealand White rabbits were inseminated (Day 0post-coitum) and exposed by dietary exposure to 0, 1000, 3000 and 11000 ppm (equivalent to 0, 41, 113 and 367 mg/kg body weight/day respectively) from Days 7 to 29post-coitum. The study consists of Part I and II. Dietary concentrations were analytically verified.

Possibly stress from construction work interfered with the performance of the study, resulting to very low pregnancy rates, including in the controls, which required additional groups of animals (part II) to be dosed. In the study report the data is reported as one pooled group as well as separately for each cohort.

 

Rabbits dosed with 11000 ppm p-TSA showed evidence of toxicity with reduced body weight which remained lower than controls for the entire treatment period. Body weight gain at 11000 ppm was also reduced at necropsy after correction for uterus weight. Transient effects on body weight were noted at 3000 ppm during the first days of dosing, but this is not considered as adverse effect in this evaluation. No treatment related toxicity was observed at 1000 ppm.

Based on the results obtained in this prenatal developmental toxicity study, it is concluded that dietary administration of p-TSA to pregnant rabbits during the period of organogenesis at a high dose level of 11000 ppm was associated with a statistically significant increase in the mean litter proportion of vertebral anomalies with or without associated rib anomaly. However, whether this was related to p-TSA or to a combination of p-TSA with incidental stress which occurred during the first part of the study could not be determined, since the findings were not observed in any of the Part II animals in this study, or in animals dosed up to 20000 ppm in the dose ranging study. No test substance related findings on foetal morphology occurred at dose levels of 1000 and 3000 ppm.

Therefore, a dosage level of 3000 ppm (113 mg/kg body weight/day in rabbits) was considered to be the No Observed Adverse Effect Level (NOAEL) for embryo/foetal developmental toxicity. Furthermore, based on the effects on body weight, food and water consumption, the maternal No Observed Adverse Effect Level (NOAEL) for p-TSA was established as being 3000 ppm (113 mg/kg body weight/day).

 

70-55-3, Toxicity to reproduction OECD422, oral-rat with p-TSA, MHW Japan, 1994

(Reported under toxicity for reproduction) Additionally there is a study screening for reproduction toxicity according to OECD 422 Combined Repeat Dose and Reproductive/Developmental Toxicity Screening Test in rat by gavage under GLP. This study included evaluation of offspring for visceral and skeletal malformation indicating no teratogenic effects of 4-methylbenzenesulfonamide.

 

70-55-3, Toxicity to reproduction 2-generation, oral- rat with p-TSA, NOTOX 474064, 2007

(Reported under toxicity for reproduction) The available full two-generation study according to OECD 416 in rats by dietary admixture concluded to a developmental toxicity related to decreased body weights of pups at 10000 ppm, accompanied by organ weight changes for the brain, spleen and thymus and a delay in vaginal opening and balano-preputial separation.Consequently, the NOAEL for developmental toxicity was established at 3000 ppm (corresponding to 165-237 mg/kg bw/day for males and 232-499 mg/kg bw/day for females).  Justification for selection of Effect on developmental toxicity: via oral route: Only available high quality study evaluating developmental toxicity

These results were used for read-across to Tosylchloramide Sodium.

 

Justification for classification or non-classification

Toxicity to reproduction has been sufficiently evaluated in studies addressing both fertility and foetal development. Available studies for the evaluation of p-TSA for these endponts indicated no concerns with respect to hazards related to reproduction, and thus no classification is required.Based on the low lipophilic (Pow = 0.6) aspect of the substance, the quick absorption and subsequent excretion in urine, accumulation of high levels into breast milk is not likely. Also the 2-generation study with test substance p-TSA shows no indication for a concern for effects on or via lactation.

Based on the read across results of the 2 -generation study in rats (OECD 416) with test substance p-TSA and the read-across results of the teratogenicity study in Rats (OECD 414) with test substance o/p-Toluenesulfonamide, the substance Tosylchloramide sodium (Chloramine-T) does not to be classified for reproductive toxicity and specific target organ toxicity - repeated exposure (STOT-RE) in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).